U.S. patent application number 17/212528 was filed with the patent office on 2021-08-12 for method for continuous second-generation ethanol production in simultaneous saccharification and co-fermentation process.
The applicant listed for this patent is Department Of Biotechnology, Indian Oil Corporation Limited. Invention is credited to Ravi Prakash GUPTA, Anshu Shankar MATHUR, Suresh Kumar PURI, Sankara Sri Venkata RAMAKUMAR, Ajay Kumar SHARMA, Ajit SINGH, Manas Ranjan SWAIN, Deepak TULI.
Application Number | 20210246474 17/212528 |
Document ID | / |
Family ID | 1000005580358 |
Filed Date | 2021-08-12 |
United States Patent
Application |
20210246474 |
Kind Code |
A1 |
SHARMA; Ajay Kumar ; et
al. |
August 12, 2021 |
METHOD FOR CONTINUOUS SECOND-GENERATION ETHANOL PRODUCTION IN
SIMULTANEOUS SACCHARIFICATION AND CO-FERMENTATION PROCESS
Abstract
The present invention relates to a process for continuous
production of second-generation ethanol from lignocellulosic
biomass via continuous simultaneous saccharification and
co-fermentation (SSCF) process, wherein the process includes a
first fermentor vessel for selectively fermenting C5 sugars and
then Continuous transferring the fermented biomass to a second
fermentor vessel for hydrolyzing the fermented biomass and then
Continuous transferring the hydrolysate to a third fermentor vessel
for selectively fermenting C6 sugars to obtain ethanol. Overall,
the ethanol yield achieved was up to 70% for both C5 and C6 sugars
from pretreated biomass; and C5 utilization exceeded 95% after
SSCF.
Inventors: |
SHARMA; Ajay Kumar;
(Faridabad, IN) ; SWAIN; Manas Ranjan; (Faridabad,
IN) ; SINGH; Ajit; (Faridabad, IN) ; MATHUR;
Anshu Shankar; (Faridabad, IN) ; GUPTA; Ravi
Prakash; (Faridabad, IN) ; TULI; Deepak;
(Faridabad, IN) ; PURI; Suresh Kumar; (Faridabad,
IN) ; RAMAKUMAR; Sankara Sri Venkata; (Faridabad,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Indian Oil Corporation Limited
Department Of Biotechnology |
Mumbai
New Delhi |
|
IN
IN |
|
|
Family ID: |
1000005580358 |
Appl. No.: |
17/212528 |
Filed: |
March 25, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
17075486 |
Oct 20, 2020 |
|
|
|
17212528 |
|
|
|
|
16351045 |
Mar 12, 2019 |
|
|
|
17075486 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 7/10 20130101; C12P
7/14 20130101; C12P 7/065 20130101 |
International
Class: |
C12P 7/06 20060101
C12P007/06; C12P 7/10 20060101 C12P007/10; C12P 7/14 20060101
C12P007/14 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 2018 |
IN |
201821008982 |
Claims
1. A process for continuous production of a second-generation
ethanol from a lignocellulosic biomass comprising; (i) adding
slurry of a pre-treated lignocellulosic biomass comprising C5 and
C6 sugars in a first fermenting vessel of a fermentor system for a
first fermentation process; (ii) Fermenting mainly C5 sugars by
incubating the pretreated lignocellulosic biomass with a cellulase
enzyme, a co-fermenting microorganism and a nutrient to obtain
ethanol; (iii) Continuous transferring fermented biomass of the
first fermenting vessel to a second fermenting vessel of the
fermentor system for conducting a hydrolysis reaction at
48-55.degree. C. for a period of 28-30 hour; (iv) transferring
hydrolysate of the second fermenting vessel to a third fermenting
vessel of the fermentor system for a second fermentation process
for 8-10 hour; (v) Fermenting mainly C6 sugars to obtain
ethanol.
2. The process as claimed in claim 1, wherein the C5 sugar is
selected from xylose and C6 sugar is selected from glucose.
3. The process as claimed in claim 1, wherein the concentration of
the cellulase enzyme in a range of 1.8-2.5 FPU/TS is employed for
the fermentation process.
4. The process as claimed in claim 1, wherein the fermentation of
C5 sugar is carried out at a temperature in a range of
33-35.degree. C. for 16-20 hour.
5. The process as claimed in claim 1, wherein the fermentation of
C6 sugar is carried out at a temperature in a range of
35-37.degree. C. for 08-10 hour.
6. The process as claimed in claim 1, wherein the pre-treated
lignocellulosic biomass slurry is added in the first fermenting
vessel of the fermentor system of step (i) without any
detoxification.
7. The process as claimed in claim 1, additionally comprising
adjusting pH of the slurry of step (i) to 5-5.5 with a pH
adjuster.
8. The process as claimed in claim 7, wherein the pH adjuster is
selected from aqueous ammonium solution, NaOH, KOH, CaCO.sub.3 or a
substance which is alkaline in nature and increases pH.
9. The process as claimed in claim 1, wherein the nutrient is
MgSO.sub.4 or any other magnesium salt. Nitrogen source such as
urea, ammonium sulfate etc is required in case pH adjuster is other
than aqueous ammonia.
10. The process as claimed in claim 1, wherein the cellulase enzyme
is from fungal or bacterial origin, composed of cellobiohydrolase
(I, II), endo-glucanase and .beta.-glucosidase along with other
accessory enzyme, wherein the other accessory enzyme is selected
from xylanase, .beta.-xyloxidase, arabinofuranosidase, and pectinse
or any other enzyme which hydolyze glucan and/or xylan.
11. The process as claimed in claim 1, wherein the co-fermenting
microorganism is selected from Saccharomyces cerevisiae, or any
ethanogenic co-fermenting microorganism such as Pichia sp., Candida
sp., and E. coli.
12. The process as claimed in claim 1, wherein the lignocellulosic
biomass is selected from straw, wheat straw, rice straw, sugarcane
bagasse, cotton stalk, barley stalk, bamboo or any agriculture
residues which contain cellulose or hemicellulose or both.
13. A process for continuous production of a second-generation
ethanol from a lignocellulosic biomass comprising: (i) adding a
slurry of pre-treated lignocellulosic biomass comprising C5 and C6
sugars with 20-22 weight % total solids (TS) and without any
detoxification in a first fermenting vessel of a fermenter system
for a first fermentation process; (ii) adjusting pH of the slurry
of step (i) to 5-5.5 with aqueous ammonium solution to obtain a pH
adjusted slurry; (iii) fortifying the pH adjusted slurry with
MgSO.sub.4 in amount of 03 g/l, along with a cellulase enzyme and a
co-fermenting microorganism; (iv) adding water to the slurry of
step (iii) to maintain 15-20 weight % TS in the slurry; (v)
incubating the slurry of step (iv) at 33-35.degree. C. for 16-20
hours for a selectively fermenting mainly C5 sugars to obtain
ethanol; (vi) Continuous transferring fermented biomass of the
first fermenting vessel to a second fermenting vessel of the
fermentor system for conducting a hydrolysis reaction at
48-55.degree. C. for a period of 28-30 hours; (vii) Continuous
transferring hydrolysate of the second fermenting vessel to a third
fermenting vessel of the fermentor system for a second fermentation
process for at 35-37.degree. C. for 08-10 hours; (viii) Fermenting
mainly C6 sugars to obtain ethanol.
14. A system for continuous production of a second-generation
ethanol from a lignocellulosic biomass, said system comprising: a
first fermentor vessel with size of 16000 M.sup.3 and hydraulic
reaction time (HRT) of 16 hours and dilution rate maintained at
0.0625 h.sup.-1; a second fermentor vessel with size of 30000
M.sup.3 and HRT of 30 hours and dilution rate maintained at 0.033
V; and a third fermentor vessel with size of 10000 M.sup.3 and HRT
of 10 hours; wherein three fermentor vessels are arranged in a
sequential manner; and wherein in-out flow rate to all the
fermentor vessels is maintained at a constant rate of 1000M.sup.3
to achieve steady state.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present patent application is a patent of addition of
the main Indian Patent Application No. 201821008982 of Filing date
Mar. 12, 2018, and Publication date Jan. 3, 2020. The present
application comprises an improvement in or a modification of the
invention claimed in the specification of the main patent applied
for in the Indian Patent Application No. 201821008982.
FIELD OF THE INVENTION
[0002] The present invention relates to a process for continuous
production of second-generation ethanol from lignocellulosic
biomass in simultaneous saccharification and co-fermentation (SSCF)
process. More particularly, the process achieves overall ethanol
yield up to 70% for both C5 and C6 sugars from pretreated biomass.
C5 utilization exceeds 95% after SSCF. This involves using three
separate vessels/fermentors for the fermentation process based on
the temperature changes required.
BACKGROUND OF THE INVENTION
[0003] Simultaneous Saccharification and
fermentation/co-fermentation (SSF/SSCF) removes sugar inhibition on
enzymatic hydrolysis thus increasing the hydrolysis sugar yield and
reducing contamination risk. Moreover, SSF/SSCF reduces the overall
reaction time and reactor volume (Kristensen et al., 2009).
SSF/SSCF sacrifices the optimal conditions for both enzymatic
hydrolysis and fermentation. Typically, in enzymatic hydrolysis and
fermentation in SSF system the temperature is kept at 37-42.degree.
C. as a compromise for better enzymatic hydrolysis and fermentation
(Dien et al., 2003b). In addition, SSF/SSCF introduces a new
inhibitor (ethanol) for enzymatic hydrolysis. But the inhibitory
effect from ethanol is much lower compared to cellobiose or glucose
(Taherzadeh & Karimi, 2007).
[0004] Continuous approach of SSCF is more economical and practical
at demo and production level because it makes the process more
economical and less labor-intensive approach. There is a report
regarding continuous SSCF by Jin et al. 2013 for ethanol production
from Ammonia Fiber Expansion (AFEX.TM.) pretreated corn stover. In
this study, the author represented enhanced ethanol production from
pretreated corn stover by following pre-hydrolysis by fermentation.
In the approach of continuous SSCF, pre-hydrolysis is followed for
24 hours and the then the fermentation is followed in three
conjugative fermentors arranged in a bioreactor train fashion. The
flow rate in the reactors is similar.
[0005] Furthermore, the main Indian Patent Application No.
201821008982 disclosed a batch SSCF process, in which fermentation
time reduced significantly with application of low dose of
cellulase enzyme in comparison to the conventional SSCF process.
However, the present invention discloses a process in a continuous
mode which has several advantages over the main Indian Patent
Application No. 201821008982 as described below. [0006] Continuous
SSCF process reduces operational area reactor vessel in demo or
production scale. [0007] Initial viscosity of fermentation process
reduces significantly (less than 90%) which makes the process more
energy viable. [0008] Dosage of yeast to the fermentor vessel (C5
and C6) will be reduced because yeast will get sufficient time to
double its cell biomass during the fermentation process [0009]
Desired temperature required for the process will be maintained
separately which also makes the process more energy saving. [0010]
Reduced chances of contamination as sugar concentration is low,
ethanol titer and yeast concentration is higher in comparison to
batch process.
[0011] Accordingly, the present invention provides a process which
overcomes the aforesaid drawback of the prior arts. In the present
invention, overall ethanol yield was achieved upto 70% for both C5
and C6 sugars from pretreated biomass. C5 utilization exceeded 95%
after SSCF. In the current practice the C5 and C6 sugars were
targeted for fermentation in a sequence manner to achieve higher
ethanol titer at short time of fermentation and low dose of
enzyme.
SUMMARY OF THE INVENTION
[0012] Present invention relates to a process for continuous
production of second-generation ethanol from a lignocellulosic
biomass, wherein the process includes a first fermentor vessel for
fermenting mainly C5 sugars and continuous transferring the
fermented biomass to a second fermentor vessel for hydrolyzing the
fermented biomass and then continuous transferring the hydrolysate
to a third fermentor vessel for selectively fermenting C6 sugars to
obtain ethanol. The C5 and C6 sugars are targeted for fermentation
in a sequential manner to achieve higher ethanol titer at short
time of fermentation (56 hours) and low dose of enzyme. Further, in
the present invention of continuous SSCF process, overall ethanol
yield up to 70% was achieved for both C5 and C6 sugars from
pretreated biomass; and C5 utilization exceeded 95% after SSCF.
Therefore, the C5 fermentation, hydrolysis and C6 fermentation is
performed in three separate vessels at the required temperature.
The present invention ultimately reduces vessel numbers, reduction
of continuous yeast dosing to fermentation vessels, low
concentration of the xylose maintained in both fermentation vessels
throughout the process which reduces the C5 fermentation time and
initial higher viscosity problem in batch process is reduced up to
90%, which ultimately saves overall energy input for stirring and
no additional filling or emptying time in steady state.
[0013] Accordingly, present invention provides a process for
continuous production of a second-generation ethanol from a
lignocellulosic biomass comprising; [0014] (i) adding slurry of a
pre-treated lignocellulosic biomass comprising C5 and C6 sugars in
a first fermenting vessel of a fermentor system for a first
fermentation process; [0015] (ii) Fermenting mainly C5 sugars by
incubating the pretreated lignocellulosic biomass with a cellulase
enzyme, a co-fermenting microorganism and a nutrient to obtain
ethanol. After 16-20 h, process made continuous by adding
pretreated slurry to maintain HRT 16-20 h; [0016] (iii) Continuous
transferring fermented biomass of the first fermenting vessel to a
second fermenting vessel of the fermentor system for conducting a
hydrolysis reaction at 48-55.degree. C. for retention time of 28-30
hours; [0017] (iv) Continuous transferring hydrolysate of the
second fermenting vessel to a third fermenting vessel of the
fermentor system for a second fermentation process for retention
time of 08-12 hours; [0018] (v) fermenting mainly C6 sugars to
obtain ethanol.
[0019] In one of the features of the present invention, the C5
sugar is selected from xylose and C6 sugar is selected from
glucose.
[0020] In another feature of the present invention, the
concentration of the cellulase enzyme in a range of 1.8-2.5 FPU/TS
is employed for the fermentation process.
[0021] In yet another feature of the present invention, the
fermentation of C5 sugar is carried out at a temperature in a range
of 33-35.degree. C. for 16-20 hours.
[0022] In still another feature of the present invention, the
fermentation of C6 sugar is carried out at a temperature in a range
of 35-37.degree. C. for 8-10 hours.
[0023] In yet another feature of the present invention, the
pre-treated lignocellulosic biomass slurry is added in the first
fermenting vessel of the fermentor system of step (i) without any
detoxification. In another feature of the present invention, the pH
of the slurry of step (i) to 5-5.5 is adjusted with a pH adjuster.
The pH adjuster is selected from aqueous ammonium solution, NaOH,
KOH, CaCO.sub.3, or a substance which is alkaline in nature and
increases pH.
[0024] In still another feature of the present invention, the
nutrient is MgSO.sub.4 or any other magnesium salt. Nitrogen source
such as urea, ammonium sulfate etc is required in case pH adjuster
is other than aqueous ammonia.
[0025] In still another feature of the present invention, the
cellulase enzyme is from fungal or bacterial origin, composed of
cellobiohydrolase (I &II), endo-glucanase and
.beta.-glucosidase along with other accessory enzyme, wherein the
other accessory enzyme is selected from xylanase,
.beta.-xyloxidase, arabinofuranosidase, and pectinse or any other
enzyme which hydolyyze glucan and/or xylan.
[0026] In yet another feature of the present invention, the
co-fermenting (C6 and C5 sugar) microorganism is selected from
Saccharomyces cerevisiae, or any ethanogenic co-fermenting
microorganism such as Pichia sp., Candida sp., Zymomonas mobilis
and E. coli.
[0027] In still another feature of the present invention, the
lignocellulosic biomass is selected from straw, wheat straw, rice
straw, sugarcane bagasse, cotton stalk, barley stalk, bamboo or any
agriculture residues which contain cellulose or hemicellulose or
both.
[0028] In one of the features, the present invention provides a
process for continuous production of a second-generation ethanol
from a lignocellulosic biomass comprising: [0029] (i) adding a
slurry of pre-treated lignocellulosic biomass comprising C5 and C6
sugars with 15-20 weight % total solids (TS) and without any
detoxification in a first fermenting vessel of a fermenter system
for a first fermentation process; [0030] (ii) adjusting pH of the
slurry of step (i) to 5-5.5 with aqueous ammonium solution to
obtain a pH adjusted slurry; [0031] (iii) fortifying the pH
adjusted slurry with MgSO.sub.4 in amount of 03-05 g/l, along with
a cellulase enzyme and a co-fermenting microorganism; [0032] (iv)
adding water to the slurry of step (iii) to maintain 15-20 weight %
TS in the slurry; [0033] (v) incubating the slurry of step (iv) at
33-35.degree. C. for 16-20 hours for a selectively fermenting
mainly C5 sugars to obtain ethanol; [0034] (vi) Continuous
transferring fermented biomass of the first fermenting vessel to a
second fermenting vessel of the fermentor system for conducting a
hydrolysis reaction at 48-55.degree. C. for a period of 28-30
hours; [0035] (vii) Continuous transferring hydrolysate of the
second fermenting vessel to a third fermenting vessel of the
fermentor system for a second fermentation process for at
35-37.degree. C. for 8-10 hours; [0036] (viii) Fermenting mainly C6
sugars to obtain ethanol.
[0037] In one of the preferred features, the present invention
provides a system for continuous production of a second-generation
ethanol from a lignocellulosic biomass, said system comprising: a
first fermentor vessel with size of 16000 M.sup.3 and hydraulic
reaction time (HRT) of 16 hours and dilution rate maintained at
0.0625 h.sup.-1; a second fermentor vessel with size of 30000
M.sup.3 and HRT of 30 hours and dilution rate maintained at 0.033
h.sup.-1; and a third fermentor vessel with size of 10000 M.sup.3
and HRT of 10 hours; wherein three fermentor vessels are arranged
in a sequential manner; and wherein in-out flow rate to all the
fermentor vessels is maintained at a constant rate of 1000M.sup.3
to achieve steady state.
BRIEF DESCRIPTION OF DRAWINGS
[0038] FIG. 1 illustrates schematic presentation of the batch and
continuous SSCF fermentation process;
[0039] FIG. 2 illustrates continuous SSCF process for ethanol
production;
[0040] FIG. 3 illustrates results of the ethanol produced in
continuous fermentation process wherein Fermentor 1 (F1): Xylose
(C5) fermentation, Fermentor 2 (F2): Enzymatic hydrolysis and
Fermentor 3 (F3): Glucose (C6) fermentation;
DETAILED DESCRIPTION OF THE INVENTION
[0041] While the invention is susceptible to various modifications
and alternative forms, specific embodiment thereof will be
described in detail below. It should be understood, however that it
is not intended to limit the invention to the particular forms
disclosed, but on the contrary, the invention is to cover all
modifications, equivalents, and alternative falling within the
scope of the invention as defined by the appended claims.
Definition
[0042] For the purposes of this invention, the following terms will
have the meaning as specified therein: "Pre-treated biomass" or
"Pretreatment of biomass" used herein clears away physical and
chemical barriers that make native biomass recalcitrant and exposes
cellulose for better enzymatic hydrolysis. In most of the
pretreatment, chemical (acid or alkali) and physical (high
temperature or pressure) parameters are used individually or in
mixed manner to remove barriers for enzymatic hydrolysis and
improve the enzymatic digestibility.
[0043] "Detoxification" used herein is the process where the
inhibitors (toxic compound such hydroxymethyl furfural, furfural,
acetic acids, formic acids, etc.) produced during the pretreatment
process are removed or neutralized from pre-treated biomass by
chemical, physical, or biological process.
[0044] "Cellulase enzyme" used herein is a mixed form of enzyme
which is mostly composed of cellobiohydrolase (I &II),
endo-hydrolase and .beta.-glucosidase. This enzyme was mostly
produced from fungal sources. Cellulase breaks down the cellulose
molecule into monosaccharide and shorter polysaccharides or
oligosaccharides. In the present invention the cellulase enzyme is
selected from commercially available cellulase enzymes which are
suitable for the purposes.
[0045] "C5 sugars" used herein represents xylose.
[0046] "C5 fermentation" used herein is xylose fermentation into
ethanol.
[0047] "C6 sugar" used herein represents glucose.
[0048] "C6 fermentation" used herein is glucose fermentation into
ethanol.
[0049] "Nutrient" used herein is MgSO.sub.4. In the salt MgSO.sub.4
used in fermentation where, Mg.sup.+2 acts as an essential enzyme
cofactor and acts as key structural component of most biological
pathways. During fermentation Mg.sup.+2 plays a major role for
proper functioning of fermenting enzymes in yeast.
[0050] Simultaneous saccharification and co-fermentation (SSCF) is
a promising strategy for obtaining high ethanol yield. This process
operates in a single fermentor vessel where the required
temperature keeps changing during the fermentation practice. But
shifting of temperature from higher to lower in a batch process
makes the process more energy intensive at higher operation level
(demo or production scale). Also, large scale plant reactors would
require transferring volume from one vessel to another vessel of
different temperatures, which would require additional emptying and
filling time. This would result in higher capital and operational
cost to the process. To overcome this issue, in the present
invention, the temperature changes (33.degree. C. for C5
fermentation, 50.degree. C. for hydrolysis and 35.degree. C. for C6
fermentation) for fermentation process are performed in three
separate fermentor vessels sequentially at their desired
temperatures. This makes the process easier, however, at large
scale operation two bioreactor train system (i.e., three vessels in
each train in a parallel manner, in total six vessels required)
arrangements are required to make the process continuous.
Continuous fermentation process reduces vessel number, reduction of
continuous yeast dosing to fermentation vessel, low concentration
of xylose maintained in both fermentation vessels throughout the
process which reduces the C5 fermentation time and initial higher
viscosity problem in batch process is reduced up to 90%, which
ultimately saves overall energy input for stirring. In the process
of the present invention, the C5 and C6 sugars targeted for
fermentation in sequence manner to achieve higher ethanol titer at
short time of fermentation and low dose of enzyme. An overall
ethanol yield up to 70% was achieved for both C5 and C6 sugars from
pretreated biomass. C5 utilization also exceeded 95% after
SSCF.
[0051] The present invention discloses a process for continuous
production of second-generation ethanol from lignocellulosic
biomass in SSCF process using three separate vessels/fermentors for
the fermentation process based on the temperature changes
required.
[0052] A continuous SSCF process as disclosed in the present
invention when compared to a batch SSCF process (see FIG. 1). In
this continuous SSCF approach three bioreactors are attached
conjugative as described in FIG. 1. The batch SSCF process and
continuous SSCF process are represented in yellow and red colored
line, respectively. In (pretreated biomass) and out (fermented
broth) flow (presented in arrow line) of the process are in
continuous mode. When all vessels in the described continuous SSCF
process reached to the designated volume then the flow rate to the
entire reactor maintained in a constant rate. The improvements of
the continuous SSCF over batch SSCF are given below in Table 1.
TABLE-US-00001 TABLE 1 Improvements of the Continuous SSCF over
Batch SSCF Continuous SSCF Batch SSCF Remark Reactor Volume 16000 +
30000 + 10000 = (12000 + 12000 + 12000) * 2 = Less working volume
56000 M.sup.3 72000 M.sup.3 reduces final cost to the process
Viscosity >1000 cP Very high (Initial 1 lakh Low viscosity cP)
improves fermentation mass flow and electric saving Yeast Dozing
Not required after Every batch requires One time yeast pitching
pitching pitching at starting of process. This reduces yeast cost
by approximately one rupee/liter of ethanol Temperature Constant in
reactors Variable (up and down) Constant temperature in the
fermentor makes the process easier and economical for
operation.
[0053] A process for continuous production of a second-generation
ethanol from a lignocellulosic biomass (see FIG. 2) comprising:
[0054] (i) adding a slurry of pre-treated lignocellulosic biomass
comprising C5 and C6 sugars with 20-22 weight % total solids (TS)
and without any detoxification in a first fermenting vessel of a
fermenter system for a first fermentation process; [0055] (ii)
adjusting pH of the slurry of step (i) to 5-5.5 with aqueous
ammonium solution to obtain a pH adjusted slurry; [0056] (iii)
Fortifying the pH adjusted slurry with MgSO.sub.4 in amount of 03
g/l, along with a cellulase enzyme and a co-fermenting
microorganism; [0057] (iv) adding water to the slurry of step (iii)
to maintain 15-20 weight % TS in the slurry; [0058] (v) incubating
the slurry of step (iv) at 33-35.degree. C. for 16-20 hours for a
fermenting mainly C5 sugars to obtain ethanol; [0059] (vi)
Continuous transferring fermented biomass of the first fermenting
vessel to a second fermenting vessel of the fermentor system for
conducting a hydrolysis reaction at 48-55.degree. C. for a period
of 28-30 hours; [0060] (vii) Continuous transferring hydrolysate of
the second fermenting vessel to a third fermenting vessel of the
fermentor system for a second fermentation process for at
35-37.degree. C. for 8-10 hours; [0061] (viii) Fermenting mainly C6
sugars to obtain ethanol.
[0062] In accordance with the present invention, steady state
ethanol production achieved using dilute acid pretreated rice
straw. In this process, three reactors of different volume are in
series with constant flow of slurry. However, different HRT
maintained by different volume size of reactor. First reactor
utilized for preferential xylose fermentation at 33.degree. C.
followed by hydrolysis in second reactor at 50.degree. C.
Thereafter, slurry moved to third reactor for mainly glucose
fermentation at 37.degree. C. This resulted into several advantages
which are tabulated in Table-1.
[0063] In another feature of the present invention, when all
vessels in the described continuous SSCF process reached to the
designated volume then the flow rate to the entire reactor
maintained in a constant rate. All the flow and the dilution in the
fermentor vessel are maintained as described in the FIG. 2. In FIG.
2, first fermentor/vessel represent for pentose sugar fermentation,
middle fermentor/vessel represent for enzymatic hydrolysis and
third fermentor/vessel for hexose sugar utilization. Respective
volume, flow and dilution rates are mentioned. At final
fermentation 32 g/L ethanol concentration produced in third
fermentor and continued in steady state. At the same time in other
fermentor (First and second) sugar release and ethanol
concentration are in steady state. This steady state achieved after
56 hours of the fermentation (see FIG. 3). In FIG. 3, Fermentor 1
(F1): Xylose (C5) fermentation, Fermentor 2 (F2): Enzymatic
hydrolysis and Fermentor 3 (F3): Glucose (C6) fermentation is
disclosed. This fermentation process followed the continuous
process after 55 hours of fermentation and in continuous
fermentation achieved after when the steady state is achieved in
all three fermenters.
Example 1
[0064] Process for Continuous Production of Second-Generation
Ethanol from a Lignocellulosic Biomass:
[0065] Pretreated biomass (slurry, TS approximately 20-22%) without
any detoxification is introduced directly to the first fermentor
vessel of the fermentor system. The pH of the slurry was adjusted
to 5-5.5 with aqueous ammonium solution (25% initial
concentration). The pH adjusted slurry was fortified with
MgSO.sub.4 (0.5%), cellulase enzyme (in-house enzyme/Ctec, 2.3
FPU/TS) and co-fermenting ethanologenic yeast Saccharomyces
cerevisiae (1 g dry cell biomass/100 gTS, xylose utilizing
genetically modified yeast). Required amount of water was added to
the process to maintain the final biomass concentration to 15%. The
whole process was incubated at 33.degree. C. for 16 hours for the
xylose fermentation. The fermented broth is then transferred to
second fermentor vessel of the fermentor system and is allowed for
hydrolysis at 50.degree. C. for 30 hours. The volume of the reactor
is maintained at 1.87 times higher as compared to pentose sugar
utilizing fermentor vessel for giving hydraulic reactor time of 30
hours. After the hydrolysis, the hydrolysate is transferred to
third fermentor vessel of a fermenting system and for the hexose
sugar fermenting vessel for 10 hours. All reactors have 1000
M.sup.3 flow. When all vessels in the described continuous SSCF
process reached to the designated volume then the flow rate to the
entire reactor was maintained in a constant rate. At final
fermentation, 32 g/L ethanol concentration was produced in third
fermentor vessel and continued in steady state. At the same time in
other fermentor (First and second) vessels, sugar release and
ethanol concentration are in steady state. This steady state was
achieved after 56 hours of the fermentation. The results of this
experiment are represented by FIG. 2.
Example 2
[0066] Process for Batch Production of Second-Generation Ethanol
from a Lignocellulosic Biomass (Main Indian Patent Application No.
201821008982)
[0067] The pH of the pretreated slurry was adjusted to 5.5 with
aqueous ammonium solution (25% initial concentration). The pH
adjusted slurry was fortified with 3 g/l MgSO.sub.4, cellulase
enzyme (Commercial enzyme, 3.3 FPU/TS) and co-fermenting
Saccharomyces cerevisiae (1 g dry cell biomass/litre, xylose and
glucose utilizing yeast). Required amount of water was added to the
process to adjust the final biomass concentration to 20%. The whole
process was incubated at 30.degree. C. for 16 h for the
fermentation with 200 rpm. When the free xylose concentration in
the slurry comes near to 6-7 g/1, the temperature of the process
was increased to 33.degree. C. and 35.degree. C., incubated for 2 h
in each temperature for better hydrolysis and fermentation. After
that temperature increased to 48.degree. C. This step mainly
required for rapid releases of glucose sugar from cellulose which
converted simultaneously with hydrolysis to ethanol by yeast
biomass. As the temperature was reached at desired target the
process was allowed to maintain the required temperature
(48.degree. C.) for 23 h for better enzymatic hydrolysis. After
this incubation the system was allowed to cool down to temperature
35.degree. C. A second dose of co-fermenting S. cerevisiae (1 g dry
cell biomass/liter) was inoculated to the system for the second
stage of fermentation. The second fermentation was stopped after 6
h of fermentation. This process took 46 h incubation including
fermentation and enzymatic hydrolysis.
[0068] In the present invention, the continuous SSCF approach is
considered as advantageous over the conventional SSCF and batch
SSCF due to several reasons as described in Table-1.
REFERENCES
[0069] 1. Krishnan, C., Sousa, L. D., Jin, M. J., Chang, L. P.,
Dale, B. E., Balan, V. 2010. Alkali-based AFEX Pretreatment for the
conversion of sugarcane bagasse and cane leaf residues to ethanol.
Biotechnology and Bioengineering, 107(3), 441-450. [0070] 2. Dien,
B. S., Cotta, M. A., Jeffries, T. W. 2003. Bacteria engineered for
fuel ethanol production: current status. Applied Microbiology and
Biotechnology, 63(3), 258-266. [0071] 3. Taherzadeh, M. J., Karimi,
K. 2007. Enzyme-based hydrolysis processes for ethanol from
lignocellulosic materials: a review. Bioresources, 2(4), 707-738.
[0072] 4. Lau, M. W., Dale, B. E. 2009. Cellulosic ethanol
production from AFEX-treated corn stover using Saccharomyces
cerevisiae 424A (LNH-ST). Proceedings of the National Academy of
Sciences of the United States of America, 106(5), 1368-1373. [0073]
5. Jin M., Gunawan C., Balan V., Yu X., Dale B. E. 2013. Continuous
SSCF of AFEX.TM. pretreated corn stover for enhanced ethanol
productivity using commercial enzymes and Saccharomyces cerevisiae
424A (LNH-ST). Biotechnology and Bioengineering, 110, 5,
1302-1311.
* * * * *